The central goal of this Program Project application is to address the central mechanism of human structural brain defects (SBD), utilizing the unique strengths of the Principal Investigators and the new breakthroughs in genetics and modeling. This Program Project Grant Application is designed to advance biomedical knowledge and make a high impact on our understanding of the basis of SBDs across the evolutionary scale, with the purpose of advancing our ability to diagnose and treat disease. In our preliminary data, we have established: 1] A broad database consisting of over 1500 human families with structural brain defects, highly enriched for first cousin consanguinity with multiple affected members. 2] Extensive development of mouse lines with neural-specific expression of Cre-recombinase. 3] A broad array of tools to study gene requirements in zebrafish development. 4] A proven track record of utilization of these unique reagents to study mechanisms of disease. As a result of our preliminary data, we have formulated this Program Project Grant Application with a two-fold thrust: 1] By taking advantage of the technical revolution in DNA sequencing and genetic engineering, we will uncover new causes of disease in humans. 2] By comparing phenotypes across these unique systems, each with its own strength/weakness we will enhance our understanding of the basic mechanisms of SBD. Since all Principal Investigators in this Program Project Grant Application have evidence for considering cell polarity as central to the developmental mechanisms of SBD, each Project has a focus on investigating cell polarity within the spectrum of the proposed Aims. Two Cores will be essential to the Program Project Grant Application since they will carry out essential functions of the Program Project Grant Application and benefit each Project: 1] Next-generation Sequencing Core to uncover new genetic causes of SBDs in each species. 2] Bioinformatics Core will provide essential functions to provide experimental design and analytical services with one-on-one training and data management and custom computational solutions. Specific Aims of the Program Project Grant Application are: 1] To uncover a host of new developmental causes of SBD from this unique human DNA resource, as well as from mutagenized mice and zebrafish. 2] To explore cell-type specificities of disease and pathogenic mechanisms of SBDs using mice and zebrafish models. 3] To utilize newly uncovered mice and zebrafish genes involved in SBDs for analysis in this human population. We believe that this Program Project Grant Application will have a major impact on our understanding of the cellular and molecular mechanisms that underlie a variety of SBDs, fully taking advantage of new breakthroughs in genomics technologies, which will set the stage for improved diagnosis and treatment. PUBLIC HEALTH RELEVANCE: This Program Project Grant Application is focused on understanding how the human brain develops and the genes that regulate its structural specificities. By utilizing inbred families, we will make maximal use of next-generation sequencing technologies, with the highest likelihood of uncovering mechanisms of development common across the evolutionary scale. OVERALL PROGRAM: Structural brain diseases (SBDs) are conditions that cause defects in the structure or volume of brain components as evidenced from a brain MRI. The program is well conceived, starting with the identification of mutations in a human cohort (Project 1) and moving to model organisms to explore the functions of these newly discovered mutations (Projects 2 and 3). This type of interaction among human and model organism biologists is unlikely to happen without the mechanism of a program project such as the one proposed. The three Projects interrelate remarkably well. The discovery of genes from the human SBD cohorts leads to modeling with morpholinos and mutagenesis in zebrafish and in knockout mice. The interdisciplinary backgrounds of the group complement each other nicely, and members of the group already have a record of collaborations, which bodes well for the success of this program. 1.Significance of the overall program and its potential to advance scientific knowledge: Strengths * Building the brain is complicated, and so a large fraction of birth defects involve the central nervous system. This creates a personal and societal health problem for newborns and children and their families. * The investigators estimate CNS problems cause 5-10% of total health care costs. Weaknesses * None 2. Investigator(s) (Qualifications of the Principal Investigators in Program Leadership and/or in Directing Individual Projects or Cores: Strengths * Gleeson,Joseph G., M.D., Principal Investigator, is Head of the Neurogenetics Laboratory at UCSD with substantial experience in brain diseases. * Bafna, Vineet, Ph.D., Principal Investigator Core C, is Professor, Computer Sciences and Engineering at UCSD. * Chi, Niel C., Co-Invest. Project 3, (Assistant Professor of Medicine, Cardiology Division, University of California, San Diego) has a primary interest in excitable tissues of the heart. He is an expert in zebrafish genetics and development and has developed transgenic lines for the investigation of cardiac and brain. He is experienced in the carrying out of genetic screens such as the one being proposed. He received the NIH Director's New Innovator Award for 2010. He is well suited to perform the proposed studies. * Frazer, Kelly A., Ph.D., Principal Investigator Core B, is Professor of Pediatrics Director, Division of Genome Information Sciences at UCSD. He has made substantial contributions to genomics including the HapMap Phase II project and developing new methods for interpreting genome wide association studies. His help is required for the proposed mapping studies. * Harismeny, 0., Ph.D., Proj. Sci. Core B, is Assistant Project Scientist, Department of Pediatrics, UCSD, is an expert in evaluating next generation sequencing data and will be invaluable for the generation of the targeted sequencing data. * Mueller, Ulrich, Ph.D. Co-Invest. Project 3, is Professor and Director, Dorris Neuroscience Center, Scripps Research Institute, and has extensive experience with mouse genetics necessary for testing the functions of the genes identified in Project 1. Weaknesses * None. 3. Innovation (Applying Novel Concepts and Innovative Approaches to the Overall Program and Collaborations): Strengths * The closeness of working collaboration between human, mouse, and zebrafish biologists is rare and will be hugely beneficial for discovering new genes and new functions. * The collection of consanguineous human families is an important resource for exploitation to discover the genetic bases for SBDs. Weaknesses * None. 4. Approach (Overall Design of the Program Project and Adequacy and Quality of the Experimental Approaches Proposed in the Projects): Strengths * The use of both forward and reverse genetic methodologies will facilitate discovery. * The use of human, mouse, and zebrafish in parallel and in close communication allows the exploitation of the unique beneficial attributes of each system. * In Project 1, they exclude SBDs that are likely due to environmental causes, which allows them to focus on genetic mechanisms, and they exclude conditions in which SBDs may not be the primary problem of the disease, which allows them to focus on the most important genes for this particular organ system. * Focusing on one set of mechanisms that might lead to SBDs, the planar cell polarity mechanism, has the advantage that they may be able to investigate this mechanism in depth. Weaknesses * Focusing on one set of mechanisms that might lead to SBDs, the planar cell polarity mechanism, has the disadvantage that they may ignore other important mechanisms important for SBDs. * In Project 1, will exome sequencing of a single affected be enough to localize the mutation? There will be many regions identical by descent in each affected person. Without genetic investigation of more than one affected or unaffected siblings, how will they know which region is important? 5. Environment (Scientific, Organizational/Institutional, and Administrative Environment of the Program): Strengths * UCSD Institute for Genomic Medicine will provide helpful collaboration. Weaknesses * None. Project 1 - Genetic Dissection of Human Structural Brain Disorders from Inbred Po Description (as provided by applicant): Human structural birth defects are present in 4-5% of live births in the US, contributing to half of all pediatric hospitalizafions. Importantly, the rates are double in most of the Middle East, Central Asia and North Africa where consanguinity rates approaching 60% are the norm. Of these, Structural Brain Disorders (SBDs) are probably the single biggest component to the long-term medical complications, greatly increased morbidity and mortality. Our data demonstrate tremendous locus and genetic heterogeneity among patients with SBDs from these geographic regions, presenting both a challenge as well as an opportunity. The challenge is to derive strategies to molecularly classify patients with these diseases. The opportunity is that these populations offer the chance to identify a much fuller picture of the genes contributing to SBDs in humans. Comprehensive discovery of mutations contributing to SBDs holds great promise for advancing understanding of determinants of brain development and function, and its consequences including epilepsy, developmental delay, and motor deficits. The search for SBD genes has been hampered by the lack of well-characterized pedigrees to perform gene discovery. The ability to generate whole exome sequence (WES) from such patients only increases the need for multiplex consanguineous pedigrees for these strategies, because the validation of potentially deleterious sequence variants (PDSV) requires segregation analysis. Dr. Gleeson has collected probably the world's largest cohort of such pedigrees with SBDs over the past 10 years, which will continue in Y1-5. From these, we will perform WES on 30 probands per year in Core A, and sequence analysis in Core B. Segregation analysis in the initial family and subsequent screening in patient cohorts, both from the Gleeson lab, as well as local clinics and the massive California Birth Defects Monitoring Service will help will validate the gene's involvement in the disease. Finally, genes identified from Project 2 and 3 will be screened in the cohort using similar high-throughput re-sequencing strategies. In Project 2 and 3, animal models will be created and utilized to identify underlying cellular pathophysiology, with a focus on altered cell polarity. PUBLIC HEALTH RELEVANCE: SBDs are a major cause of epilepsy, developmental delay and intellectual disability, and can contribute to a host of neuropsychiatric disorders. We propose to use WES to discover genes that case SBDs in a highly selected cohort of 30 independent families/year negative for mutations in known genes. Synergy with the other Projects and Cores will explore the gene's prevalence in disease, and underlying pathogenesis.